Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium

Hoffmann, Stefanie; Schmidt, Christiane; Walter, Steffi; Bender, Jennifer K.; Gerlach, Roman G.

doi:10.1371/journal.pone.0172630

Cited by 52 publications

(57 citation statements)

References 69 publications

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“…Primers for generating pOPC-001, pOPC-003 and pFOK are reported in Supplementary Table S1. pOPC-001 was obtained by combining the kanamycin resistance cassette and the I-SceI restriction site from pWRG717 [30], the origin of replication (R6Kγ) and origin of transfer (oriT) from pGP704 [6, 31] and the tetR and I-sceI locus from pWRG730 [30] using Gibson assembly. pOPC-003 was generated by replacing the tetR and I-sceI locus from pOPC-001 with sacB from pEXG2 [32].…”

Section: Methodsmentioning

confidence: 99%

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

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11We describe a versatile method for chromosomal gene editing based on classical consecutive 12 single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a 13 convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector 14 resolution. We used this method to generate in frame deletions, insertions and point mutations in 15Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing 16 also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates. 17 18 [19][20][21], but this strategy is more cumbersome for bacteria with limited recombination activities 34 [22][23][24]. 35Here, we combined several improvements for establishing a time-efficient versatile 36 method for consecutive single cross-overs in multiple bacterial species. We used rapid Gibson 37 assembly of PCR products [25] to generate suicide vectors with dual negative selection with I-38SceI and SacB [26, 27] (Fig 1a), which increased counter-selection efficiency to 100% for nearly 39 all tested deletions, insertions and point mutations. We employed an E. coli donor strain that 40 simplifies donor removal after conjugation and avoids common problems with contaminating 100 mM (Sigma Aldrich C1016-500G) containing 15% of glycerol (AppliChem, A1123,1000). 57 Bacteria were resuspended in 5 ml ice-cold CaCl2 100 mM containing 15% of glycerol and 200 µl 58 aliquots were frozen and stored at -80°C. Super-Optimal broth with Catabolite repression (SOC) 59 (tryptone 20 g/L, yeast extract 5 g/L, NaCl 0.5 g/L, KCl 0.186 g/L, MgSO4 4.8 g/L and glucose 3.6 60 g/L) medium was used after heat shock. Kanamycin (Roth T832.4) at a final concentration of 50 61 µg/ml and gentamicin (Gibco 15750-037) at a final concentration of 15 µg/ml were used to select 62 E. coli transformants. For positive selection, kanamycin (Roth T832.4) at a final concentration of 63 50 µg/ml, gentamicin (Gibco 15750-037) at a final concentration of 30 µg/ml, or potassium tellurite 64 (Sigma P0677) at a final concentration of 10 µg/ml, were used to select for S. typhimurium, P.

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Section: Methodsmentioning

confidence: 99%

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

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“…Wildtype Salmonella enterica serovar Typhimurium ( S. typhimurium ) SJW1103 is motile (41). The Δ fliT ::K m R mutant in which the fliT gene was replaced by a kanamycin resistance cassette was constructed using the λ Red recombinase system (42). Strains containing chromosomally encoded FliT or FlgN variants were constructed by aph -I-SceI Kanamycin resistance cassette replacement using pWRG730 (42).…”

Section: Methodsmentioning

confidence: 99%

“…The Δ fliT ::K m R mutant in which the fliT gene was replaced by a kanamycin resistance cassette was constructed using the λ Red recombinase system (42). Strains containing chromosomally encoded FliT or FlgN variants were constructed by aph -I-SceI Kanamycin resistance cassette replacement using pWRG730 (42). Bacteria were cultured at 30–37°C in Luria-Bertani (LB) broth containing, where appropriate, ampicillin (100 mg/ml) or chloramphenicol (20 mg/ml) and collected by centrifugation (6,000 g for 10 min).…”

Section: Methodsmentioning

confidence: 99%

Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion

Bryant¹,

Chung²,

Fraser³

2020

Preprint

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23 24 Keywords 25 bacterial flagella biogenesis; Type III Secretion System; proton motive force; 26 protein export 27 28 Abstract 29 Bacterial flagella are assembled from thousands of protein subunits that are unfolded 30 and exported across the cell membrane by a specialized flagellar Type III Secretion 31 System (fT3SS). Export of subunit cargo is fuelled by the proton motive force (pmf), 76 (FlgN binds FlgK and FlgL, FliT binds FliD, and FliS binds FliC) (4-6). The 77 chaperones then pilot their cognate subunits to the specialized flagellar Type III 78 secretion system (fT3SS) machinery at the base of each flagellum (7-11).79 Chaperone-subunit complexes initially dock at the FliI component of the flagellar 80 export ATPase, which is evolutionarily related to the F1-ATPase (12). Chaperoned 81 subunits are then thought to interact with the integral membrane export gate 82 component FlhA, where chaperones are released and the subunit cargo is unfolded 83 for translocation across the inner membrane, via the FlhAB-FliPQR export gate, into 84 a narrow central channel in the growing flagellum (9-13). Once released from their 85 cognate subunit cargo, the unladen chaperones FlgN and FliT -but not the flagellin 86 chaperone, FliS -are recruited by the FliJ stalk component of the ATPase, which 87 transfers them to newly synthesised cognate cargo to create a local cycle of 88 chaperone-subunit binding at the membrane fT3SS (8). This is thought to promote 89 export of the minor filament subunits that form the hook-filament junction and the 90 filament cap, which are required for initiation of filament assembly (14).91 92 Export of flagellar subunit cargo across the membrane is powered by the pmf and the 93 flagellar ATPase complex(15, 16). Specifically, the ATPase is thought to facilitate 94 unfolding of subunit cargo (17). In addition, analogous to F-and V-type ATPases, 95 ATP hydrolysis is proposed to drive rotation of the ATPase stalk, FliJ, which interacts 96 with the nonameric export gate component, FlhA, converting the gate into a highly 97 efficient proton-protein antiporter that utilises the ΔΨ electric component of the pmf 98 (18). What is unclear is how FliJ activation of the export gate is regulated in the 99 absence of subunit cargo to prevent constitutive proton influx and wasteful 100 dissipation of the pmf. One way in which export gate activation could be regulated is 101 by sequestration of FliJ by other binding partners, specifically the export chaperones 102 FlgN and FliT. A role for chaperones in the regulation of T3SS activity is supported 103 by the finding that the Psuedomonas virulence T3SS chaperone PcrG binds the FliJ 104 homologue, PscO, and that loss of this interaction results in upregulation of effector 105 secretion and renders the T3SS more resistant to pmf collapse (19). These 106 observations suggest that the chaperone-FliJ interaction is central to the regulation of 107 T3SS activity, however, the mechanistic basis of this regulation remains obscure.108 109 Here, we identify point mutant ...

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“…The generalized transducing phage of Salmonella enterica serovar Typhimurium P22 HT105 / 1 int-201 was used in all transductional crosses (37). Deletions were constructed using λ-RED homologous recombination (38, 39). Medium was supplemented with 12.5 μg/ml chloramphenicol (Cm) or 100 ng/ml anhydrotetracycline (AnTc) if necessary.…”

Section: Methodsmentioning

confidence: 99%

Controlling minimal and maximal hook-length of the bacterial flagellum

Guse

Rohde

Erhardt

2020

Preprint

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22Hook-length control is a central checkpoint during assembly of the bacterial flagellum. During 23 hook growth, a 405 amino acids (aa) protein, FliK, is intermittently secreted and thought to 24 function as a molecular measuring tape that, in Salmonella, controls hook-length to 55 nm ± 6 25 nm. The underlying mechanism involves interactions of both the α-helical, N-terminal 26 domain of FliK (FliKN) with the hook and hook cap, and of its C-terminal domain with a 27 component of the export apparatus. However, various deletion mutants of FliKN display 28 uncontrolled hook-length, which is not consistent with a ruler mechanism. Here, we carried 29 out an extensive deletion analysis of FliKN to investigate its contribution in the hook-length 30 control mechanism. We identified FliKN mutants deleted for up to 80 aa that retained wildtype 31 motility. However, the short FliK variants did not produce shorter hook-lengths as expected 32 from a physical ruler. Rather, the minimal length of the hook depends on the level of hook 33 protein production and secretion. Our results thus support a model in which FliK functions as 34 a hook growth terminator protein that limits the maximal length of the hook, and not as a 35 molecular ruler that physically measures hook-length. 37Many bacteria use a complex nanomachine, called the flagellum, to propel themselves through 38 liquid environments. Flagella consist of three main structures, (i) a membrane-spanning basal 39 body complex, which is connected by (ii) a flexible hook to (iii) the long rigid filament (1). 40The hook adapts a curved structure composed of approximately 120 subunits of the same 41 protein, FlgE, reaching a length of 55 nm (± 6 nm) in Salmonella enterica (2-4). We recently 42 showed that the physiological hook-length of 55 nm is optimal for the formation of the 43 flagellar filament bundle in Salmonella, as too short hooks might be too stiff and too long ones 44 might buckle because of a too high flexibility, thereby disrupting the filament bundle also in 45 the absence of chemotactic stimuli (5). 46Hook-length control has been intensely studied over the years as unraveling the precise 47 underlying mechanism is complex. Even more so, considering that the control of hook-length 48 is directly connected with substrate specificity switching of the entire flagellar protein 49 secretion machinery (6). A type-III secretion system (T3SS) secretes most building blocks of 50 the flagellum. The primary export gate is made of FliP, FliQ and FliR, which form an unique 51 helical pore complex; and FlhA and FlhB, which make up the substrate docking platform and 52 are involved in energy transduction (7-11). Substrate specificity switching of the T3SS 53 describes the ability of this machinery to switch from recognizing early substrates, such as the 54 rod and hook subunits, to accepting and secreting late substrates, including the hook-filament 55 junction proteins, as well as the filament subunits (6, 9). This enables the cell to coordinate 56 gene expression with the spat...

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Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium

Cited by 52 publications

References 69 publications

Efficient Dual-Negative Selection for Bacterial Genome Editing

Efficient Dual-Negative Selection for Bacterial Genome Editing

Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion

Controlling minimal and maximal hook-length of the bacterial flagellum

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